Surface plasmon resonance (SPR) spectroscopy is a key enabling technology in the analysis of molecular interactions, an area of increasing importance for scientists in the academic, pharmaceutical, and biotechnology markets. SPR based analytical instruments provide real-time specificity, affinity, and kinetics information of biomolecular interactions without requiring flourophore labeling. These data give insights into protein functionality, elucidate disease mechanisms, and play a key role in the drug discovery process. SPR can monitor interactions between proteins and immobilized ligands in real time to provide specificity, affinity, and kinetics information. These data give insights into protein functionality, elucidate disease mechanisms, and play a key role in the critical decisions needed for the efficient development and production of therapeutics. SPR instruments are used by scientists in the academic, pharmaceutical, and biotechnology sectors in key application areas such as antibody characterization, proteomics, immunogenicity, lead characterization, and biopharmaceutical development and production.
Although several commercial SPR instruments are currently available, their basic designs do not differ significantly from the original concept described 25 years ago. Commercially available SPR instruments have been limited in their application to high throughput screening and proteomics analysis by the low number of channels that can be detected simultaneously. Providing robust multichannel SPR instrumentation with high sensitivity is a key challenge in the continued development of SPR spectroscopy. Multichannel SPR instrumentation with high sensitivity is needed for direct detection in high throughput screening in the search for new pharmaceuticals. Current SPR detection limits can be insufficient for the direct detection of low concentrations of small molecules, particularly those with low binding affinities. The overall value and utility of SPR spectroscopy would be greatly increased in the drug discovery process, in particular for small molecule research, with the development of commercial SPR instrumentation with multichannel performance and high sensitivity. The analysis of biomolecular interactions is an integral part of the drug discovery process, and many millions of dollars are spent early in drug development on screening compounds for receptor binding.
SPR spectroscopy has been commercialized by several companies and has become a competitive technology in the field of direct, real time observation of protein interactions. Biacore, a major provider of SPR systems, was purchased in 2006 by GE Healthcare for $390 million. The global market for protein interaction analytical systems and consumables is estimated to be $650 million.
SPR spectroscopy is an optical technique that detects changes in the refractive index in the immediate vicinity of a thin film of metal deposited on a glass substrate. The surface plasmon resonance is observed as a dip in the intensity of the reflected light from a metal film, typically gold or silver, that is in contact with a dielectric (solution). The angle of minimum intensity of reflected light, the resonance angle, is affected by changes in the refractive index of the medium near the surface of the metal film. Binding of molecules in solution to surface immobilized receptors alters the refractive index of the medium near the metal surface. By tracking the wavelength, incident angle, or intensity of the reflected light near the resonance angle, changes in the refractive index near the metal film (˜100-500 nm) can be monitored in real time to accurately measure the amount of bound analyte, its affinity for the receptor, and the association and dissociation kinetics of the interaction.
Much research effort has been focused on the development of multichannel SPR instrumentation. Multichannel detection is particularly significant for high throughput screening applications such as drug discovery and proteomics research where many thousands of ligand-receptor or protein-protein interactions must be rapidly examined. The simultaneous measurement of multiple channels has the technical advantage of allowing designated in situ reference channels that can be used to normalize for instrument errors arising from sensor inhomogeneity, uneven sample introduction, and temperature variation. Additionally, multichannel detection allows for dedicated control channels that can be used to probe signal shifts using repeated standards to improve the quality of the binding data. The current state of the art is a four-channel system based on angular interrogation made by Biacore. A significant obstacle to multichannel SPR systems is that SPR angle or wavelength detection modes are cumbersome to implement in large arrays due to the optical complexity of the instrumentation. As a result, researchers have recently focused on SPR imaging approaches because the spatial resolution afforded from imaging with a 2D detector array can be combined with patterned microarrays of biomolecules to allow for high throughput analyses. However, it is often difficult to maintain sensitivity when using SPR designs based on intensity detection.
Commercial SPR instruments are generally capable of resolving a change of refractive index within about 1×10−5 to 1×10−6, which corresponds to a mass sensitivity of ˜1 pg/mm2 of absorbed analytes, depending on the surface functionalization chemistry. This sensitivity can be insufficient for the direct detection of low concentrations of small molecules, particularly those with low binding affinities. For example, in direct immunoassays, antibodies are immobilized on the sensor surface and subjected to the binding interaction of the analyte of interest. The change in resonance angle due to the binding interaction between the analyte and the antibody is directly proportional to the concentration of bound analyte. Although straightforward to perform, direct methods are often only useful for large molecules because small molecules have insufficient mass to effect a measureable change in the refractive index. As a result, many direct immunoassay experiments involving the detection of small molecular analytes are based on fluorescence detection using labeled analytes. Although alternative assay methods such as indirect competitive inhibition can be used to enhance small molecule detection, direct detection assays are often desirable due to their simplicity. The overall value and utility of SPR spectroscopy would be greatly increased in the drug discovery process, in particular for small molecule research, with the development of commercial SPR instrumentation with multichannel performance and high sensitivity. Most traditional SPR measurements are based on the Kretschmann configuration where a prism is used to couple the light into the metal film. The surface plasmon resonance occurs when the external light energy resonantly induces the free electrons of the metal to oscillate at the metal-dielectric interface. As a result, the radiant energy is absorbed by the metal at a certain incident angle, and the resonance coupling is observed as a sharp dip in the reflected light spectrum whose angular position is extremely sensitive to the index of refraction of the dielectric medium in contact with the metal surface. Previous work to enhance SPR sensitivity has focused on narrowing the SPR resonance to increase resolution in either wavelength or angle detection modes. This has been accomplished through a variety of designs where multilayer dielectric structures are deposited on either side of the SPR active metal film. These approaches have led to sensitivity enhancements as much as seven times that of conventional SPR sensors. Similar sensitivity improvements have been made for intensity detection modes by enhancing the SPR image contrast with dark field methods and polarization contrast methods. These techniques have shown sensitivity to changes in refractive index of 2×10−6. The challenge lies in maintaining these high sensitivities with a multichannel instrument.